Accurate determination of the preferred aggregation number of a micelle-encapsulated membrane protein dimer

The structures of membrane proteins, such as the C99 dimer, are commonly resolved using protein nuclear magnetic resonance (NMR), which employs micelles as membrane mimics. Protein NMR is not able to determine the aggregation numbers of micelles, however, and, while several experimental techniques exist for quantifying the aggregation numbers of pure micelles, these are generally not applied to micelle-encapsulated proteins. Molecular-dynamics simulations of the C99 dimer and other transmembrane proteins have been performed to complement micelle-phase protein structure studies. In these cases, it has often been assumed that the aggregation number is the same as that of the pure, empty micelle. Here, using a multiscale simulation approach combining coarse-grained and all-atom models, we provide a convenient method for testing that assumption while also accounting for the finite-size effects inherent in computer simulations of micelle self-assembly. Employing large, unbiased, coarse-grained molecular-dynamics simulations of dodecylphosphocholine (DPC) surfactant and C99 protein dimer self-assembly, we determined the radius of gyration to be 21.6 ± 2.0 Å for the micelle-encapsulated dimer and 16.0 ± 1.0 Å for the pure DPC micelle. Using these radii of gyration, we performed all-atom simulations of DPC-encapsulated C99 protein dimers with preferred aggregation numbers of 100 and 54 DPCs to test the effect of using an accurate versus a naive estimate of aggregation number on the structure of the transmembrane protein dimer. Through atomistic simulations, we determined that the structure of the transmembrane protein dimer is significantly impacted by the aggregation number of the micelle. In addition, the number of surfactant defects depends on the aggregation number, which in turn impacts the extent of water penetration and the stability of the protein's secondary structure. Taken together, our results demonstrate the importance of using an accurate estimate of the aggregation number in simulations of micelle-encapsulated protein structures.